EP1495329A1 - Method for the identification of modulators of a secretase activity - Google Patents
Method for the identification of modulators of a secretase activityInfo
- Publication number
- EP1495329A1 EP1495329A1 EP02722595A EP02722595A EP1495329A1 EP 1495329 A1 EP1495329 A1 EP 1495329A1 EP 02722595 A EP02722595 A EP 02722595A EP 02722595 A EP02722595 A EP 02722595A EP 1495329 A1 EP1495329 A1 EP 1495329A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- protein
- secretase
- anyone
- activity
- invertase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
- G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
- G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
- G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
- G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
- G01N2333/96472—Aspartic endopeptidases (3.4.23)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Definitions
- Said reporter gene is preferably selected from genes such as CYH2 or CANl conferring sensitivity to a chemical .
- Any recognition sequence of a known secretase can be used for the construction of said fusion protein of the present invention.
- Preferred secretase recognition sites are selected from the ⁇ site and the ⁇ site of the human amyloid precursor protein (APP) and the S2 site of Notch 1 protein.
- a much preferred recognition site is the ⁇ site of human APP in which the Lys595Asn and Met596Leu changes were introduced (Swedish APP mutant) .
- Fig. 2B shows a schematic drawing of the in- vertase-APP reporter.
- the invertase-APP reporter was constructed by fusing the full-length invertase (aa 1-532) to a portion of APP (aa 590-695) harboring the secretase sites, the transmembrane domain and the cytoplasmic tail. Fusion of the ER retention signal DEKKMP (Seq. Id. No. 1) to the C-term further retards growth of clones that do not express an active secretase;
- Fig. 2D shows that BACE enhances growth on sucrose plates.
- the invertase-APP fusion protein harbor- ing the Swedish mutation was co-expressed with the indicated constructs [BACE, empty vector or Yap3p (a yeast secretase that specifically cleaves APP at the ⁇ -site) ] in a yeast strain deficient for the suc2 gene and the endogenous ⁇ -secretases mkc7 and yap3. Growth on sucrose plates was monitored after 2.5 days and 3 days, respectively;
- Fig. 3 shows principles of a cellular selection system to identify modulators of secretases.
- Fig. 3A-C show schematic drawings of yeast cells.
- the re- porter gene is either the LacZ or different selectable marker genes, that can act either positively or negatively on growth. These genes are under the control of the Gall/10 promoter (denoted by the GAL4 transcription factor) ;
- Fig. 3A shows default readout (without BACE) :
- the reporter gene is induced.
- Biscrel cells harboring the invertase-APP fusion protein in the absence of any secretase activity express the reporter gene when grown on 5% sucrose/2% galactose.
- Galactose activates transcription of the reporter gene, whereas sucrose is inert to the system in the absence of secreted invertase;
- Fig. 4A shows a schematic representation of the reporter gene.
- the HIS3 open reading frame (ORF) was fused to the 47 N-terminal amino acids of GAL10.
- the expression of the GAL10-HIS3 fusion protein is under the control of the GAL10 promoter, the expression of the LacZ ORF is controlled by the GAL1 promoter.
- the arrows indicate the transcription start points.
- the boxes labeled I, II, III and IV represent GAL4 binding sites;
- Fig. 6 shows a schematic drawing of the in- vertase-Notch 1 reporter.
- the invertase-Notch 1 reporter was constructed by fusing the full-length invertase (aa 1-532) to a portion of Notch 1 (aa 1714-1876) harboring the S2 site of Notch 1 protein, the transmembrane domain and the cytoplasmic tail. Fusion of the ER retention signal DEKKMP (Seq. Id. No. 1) to the C-term further retards growth of clones that do not express an active secretase and Fig.
- the GALl-10 regulatory region contains four UAS G elements located between the divergent GALl and GAL10 promoters. These UAS G elements are bound by Gal4p in a cooperative manner. Due to this cooperativity, the system is rather sensitive towards changes in the concentration of Gal4p, which is reduced 3- to 5-fold in the presence of glucose. Gal ⁇ Op significantly contributes to glucose repression of the GALl-10 genes by binding to Gal4p to mask its activation domain, thereby preventing expression of these GAL genes [6].
- the GALl promoter contains an additional regulatory element, which is the binding site for the re- pressor Miglp.
- invertase secretion of invertase enables yeast to use sucrose as a carbon source.
- the invertase hydrolyses su- crose to yield fructose and glucose.
- the endogenous gene encoding the invertase (the SUC2 gene) was knocked out.
- a recombinant invertase was fused to the N-terminus of a portion of APP harboring the transmembrane domain as well as the ⁇ - and ⁇ -sites (residues 590-695) .
- an ER retention signal was added to the C-terminus of this fusion construct (Fig. 2A, B) .
- yeast endogenous secretases described in the literature which can cleave APP at the ⁇ - site: Yap3p and Mkc7 [1 2] . Since these proteins have ⁇ - secretase activity that constitutively cleaves APP, their respective genes had to be knocked out in order to investigate ⁇ -secretase activity in yeast using the invertase reporter system described in WO 01/75068.
- a reporter construct was cloned which expresses the LacZ gene under the control of the GALl promoter and the divergently oriented HIS3 gene under the control of the GAL10 promoter. As in the case of the endogenous GALl and GAL10 genes, four UAS G elements are located between these two promoters.
- This reporter construct was integrated in yeast cells deficient for suc2 , mkc7 and yap3 to create the strain Biscrel. If this strain is grown in the presence of galactose, expression of the LacZ gene and the HIS3 gene is induced by the UAS G -binding Gal4p activator.
- BACE is inhibited, either by mutations or by an inhibiting compound, the invertase moiety re- mains anchored to the ER-membrane via the APP domain, the sucrose in the medium is not hydrolyzed, and expression of the HIS3 and LacZ genes is induced by galactose (Fig 3C) .
- Biscrel was transformed with plasmids expressing different ⁇ -secretase activities together with the invertase-APP (Sw) construct, or its wildtype variant (invertase-APP) .
- Empty plasmids and an invertase- expressing plasmid were used as negative and positive controls, respectively.
- Liquid cultures were grown in 5 % sucrose, 2 % galactose, 0.1 % glucose drop-out medium (The medium contained histidine for this assay as the cultures should grow equally) .
- the maximal level of LacZ expression in this assay was obtained with transformants harboring two empty plasmids.
- the expression of the HIS3 gene was quantified by a growth assay in liquid medium.
- Biscrel cells were cultivated in liquid -his medium containing 5% sucrose and 2% galactose (selective condi- tions) .
- Two-ml cultures were inoculated with equal amounts of cells transformed with the constructs of interest.
- Cell density was measured after 24 h.
- the same amounts of cells were used to inoculate cultures in non- selective medium.
- This control experiment showed that none of the constructs per se had an effect on cell growth under non-selective conditions (Fig. 4C, columns 1-4) .
- the cell densities measured after growth in selective medium are displayed in figure 4C, columns 5-8.
- the screening system is as well suitable to screen for modulators of other secretases .
- Figure 5 shows the results of such an experiment.
- Yap3p is a yeast secretase which cleaves APP at the ⁇ -site.
- the experimental procedures were the same as described for fig. 4B.
- the mutated Yap3p as well as the wildtype version were provided on a yeast expression vector.
- Yap3p cleaves the in- vertase-APP fusion protein at the ⁇ -site thereby liberating the invertase.
- the observed effect on LacZ expression is comparable to the effect observed when soluble and secreted invertase was expressed (last column) .
- the first column indicates the value obtained with the empty vector (0.4532 ⁇ -gal units) ; the second column indicates the value obtained for Yap3p (0.0724 ⁇ -gal units).
- the cleavage of the invertase-Notch fusion protein was confirmed by de- tecting the cleavage products in a Western blot (data not shown) .
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Toxicology (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AT02722595T ATE322019T1 (en) | 2002-04-18 | 2002-04-18 | METHOD FOR DETECTING MODULATORS OF SECRETASIS ACTIVITY |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/IB2002/001342 WO2003087842A1 (en) | 2002-04-18 | 2002-04-18 | Method for the identification of modulators of a secretase activity |
Publications (2)
Publication Number | Publication Date |
---|---|
EP1495329A1 true EP1495329A1 (en) | 2005-01-12 |
EP1495329B1 EP1495329B1 (en) | 2006-03-29 |
Family
ID=29227360
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP02722595A Expired - Lifetime EP1495329B1 (en) | 2002-04-18 | 2002-04-18 | Method for the identification of modulators of a secretase activity |
Country Status (6)
Country | Link |
---|---|
US (1) | US20060068388A1 (en) |
EP (1) | EP1495329B1 (en) |
AU (1) | AU2002253468B2 (en) |
CA (1) | CA2482824A1 (en) |
DE (1) | DE60210266T2 (en) |
WO (1) | WO2003087842A1 (en) |
Families Citing this family (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2544074A1 (en) * | 2003-10-24 | 2005-05-06 | Esbatech Ag | Method for the identification and/or validation of receptor tyrosine kinase inhibitors |
WO2006133588A1 (en) * | 2005-06-13 | 2006-12-21 | Oncalis Ag | ARYL UREA COMPOUNDS AS β-SECRETASE INHIBITORS |
EP1734039A1 (en) * | 2005-06-13 | 2006-12-20 | Esbatech AG | Aryl urea compounds as BETA-secretase inhibitors |
US20100042072A1 (en) * | 2008-08-13 | 2010-02-18 | Searete Llc, A Limited Liability Corporation Of The State Of Delaware | Biological targeting compositions and methods of using the same |
US20100040546A1 (en) * | 2008-08-13 | 2010-02-18 | Searete Llc, A Limited Liability Corporation Of The State Of Delaware | Biological targeting compositions and methods of using the same |
US20110070153A1 (en) * | 2008-08-13 | 2011-03-24 | Searete, Llc, A Limited Liability Corporation Of The State Of Delaware | Artificial cells |
US8211656B2 (en) | 2008-08-13 | 2012-07-03 | The Invention Science Fund I, Llc | Biological targeting compositions and methods of using the same |
WO2014068408A2 (en) | 2012-10-23 | 2014-05-08 | Caris Life Sciences Switzerland Holdings, S.A.R.L. | Aptamers and uses thereof |
US10942184B2 (en) | 2012-10-23 | 2021-03-09 | Caris Science, Inc. | Aptamers and uses thereof |
AU2013361323B2 (en) | 2012-12-19 | 2018-09-06 | Caris Science, Inc. | Compositions and methods for aptamer screening |
CN108004198B (en) * | 2016-10-28 | 2021-07-16 | 华中农业大学 | Method for establishing high-throughput drug screening model based on ICAM-1 signal channel |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6083693A (en) * | 1996-06-14 | 2000-07-04 | Curagen Corporation | Identification and comparison of protein-protein interactions that occur in populations |
DE19641180A1 (en) * | 1996-09-24 | 1998-03-26 | Schering Ag | A method of displaying APP secretase modulation and its use as an agent for the treatment of Alzheimer's disease |
DE19856261C1 (en) * | 1998-12-07 | 2000-03-30 | Hoechst Marion Roussel De Gmbh | Detection of gamma-secretase by detection of A-beta peptide useful for determining gamma-secretase activity and for identifying inhibitors |
DE19920514A1 (en) * | 1999-05-05 | 2000-11-16 | Boehringer Ingelheim Pharma | Methods for finding proteases that specifically cleave membrane-bound substrates |
US20020025508A1 (en) * | 2000-01-06 | 2002-02-28 | Katja Fechteler | Process for finding a protease inhibitor |
WO2001062897A1 (en) * | 2000-02-25 | 2001-08-30 | Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw | Presenilin deficient multipotent cell lines and screening methods for intramembrane regulated proteolytic activities using these lines |
US6333167B1 (en) * | 2000-03-10 | 2001-12-25 | American Home Products Corp. | Methods and reagents for identifying inhibitors of proteolysis of membrane-associated proteins |
CA2401155A1 (en) * | 2000-04-05 | 2001-10-11 | Alcide Barberis | Method for identify polypeptides with protease activity |
TWI316960B (en) * | 2000-07-19 | 2009-11-11 | Upjohn Co | Substrates and assays for beta-secretase activity |
WO2002025508A2 (en) * | 2000-09-19 | 2002-03-28 | Christophe Dutordoir | Automatic receipt confirmation system for electronic mail |
-
2002
- 2002-04-18 AU AU2002253468A patent/AU2002253468B2/en not_active Ceased
- 2002-04-18 EP EP02722595A patent/EP1495329B1/en not_active Expired - Lifetime
- 2002-04-18 US US10/512,001 patent/US20060068388A1/en not_active Abandoned
- 2002-04-18 CA CA002482824A patent/CA2482824A1/en not_active Abandoned
- 2002-04-18 WO PCT/IB2002/001342 patent/WO2003087842A1/en not_active Application Discontinuation
- 2002-04-18 DE DE60210266T patent/DE60210266T2/en not_active Expired - Fee Related
Non-Patent Citations (1)
Title |
---|
See references of WO03087842A1 * |
Also Published As
Publication number | Publication date |
---|---|
US20060068388A1 (en) | 2006-03-30 |
DE60210266D1 (en) | 2006-05-18 |
WO2003087842A1 (en) | 2003-10-23 |
AU2002253468A1 (en) | 2003-10-27 |
AU2002253468B2 (en) | 2008-04-03 |
CA2482824A1 (en) | 2003-10-23 |
DE60210266T2 (en) | 2007-01-11 |
EP1495329B1 (en) | 2006-03-29 |
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